Expression of the Csp Protein Family upon Cold Shock and Production of Tetracycline in Streptomyces aureofaciens

Expression of the Csp Protein Family upon Cold Shock and Production of Tetracycline in Streptomyces aureofaciens

Biochemical and Biophysical Research Communications 265, 305–310 (1999) Article ID bbrc.1999.1673, available online at http://www.idealibrary.com on ...

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Biochemical and Biophysical Research Communications 265, 305–310 (1999) Article ID bbrc.1999.1673, available online at http://www.idealibrary.com on

Expression of the Csp Protein Family upon Cold Shock and Production of Tetracycline in Streptomyces aureofaciens Karel Mikulı´k, 1 Quoc Khanh-Hoang, Petr Halada, Silvie Bezousˇkova´, Oldrˆich Benada, and Vladislav Beˆhal Institute of Microbiology, Academy of Sciences of the Czech Republic, Vı´denˆska´ 1083, Prague 4, 142 20, Czech Republic

Received September 28, 1999

A shift down in temperature causes in Streptomyces aureofaciens a transient repression of polypeptide synthesis. During the acclimation phase 32 proteins were synthesized. The addition of tetracycline (200 mg/ml) to cells from exponential phase of growth leads to induction of 27 novel proteins and 17 upregulated proteins migrated in 2-D gel as proteins expressed upon cold shock. Immunoblot analysis using antibodies raised against CspB, CspC, and CspD of Bacillus subtilis revealed five cross-reactive proteins of the Csp family. Proteins CspB and CspD are predominantly induced at low temperature or by the presence of tetracycline. Expression of Csp proteins during the acclimation phase is regulated on the transcription level. Proteins of the Csp family have been shown to be associated with ribosomes and can be removed by 1 M NH 4Cl. As expression of Csp proteins differs during development or temperature shift down, these proteins can be considered as trans-acting factors to form contacts with the coding region of specific mRNAs. © 1999 Academic Press

Streptomycetes are soil microorganisms, exposed to various physical and chemical stresses that activate specialized responses including synthesis of antibiotic, hydrolytic enzymes and/or morphological differentiation from vegetative cells to aerial mycelium and spores. Many stress conditions can induce the heat or cold shock response. These observations suggest that there may be multiple cellular targets or sensors that generate the inducing signal. It has been shown that a temperature shift down results in an increase in 70S ribosomes followed by increases in 30S and 50S sub1 To whom correspondence should be addressed. E-mail: [email protected] biomed.cas.cz.

units. The increase in 70S ribosomes was due to a block in initiation of translation (1). The downshift in temperature is not sole inducer of cold shock response, because antibiotics that interfere with the functions of aminoacyl acceptor site on ribosomes as chloramphenicol, tetracycline, erythromycin, spiramycin, and fusidic acid induces the response (2– 4). Cold shock causes the transient induction of proteins that affect various steps of translational system (5– 8). In E. coli protein CspA was originally described as the major cold-shock protein (9) and nine genes with high sequence similarity to CspA were identified (10). Of the nine Csp proteins in E. coli only three, CspA, CspB and CspG are cold inducible. Protein CspD is induced during the stationary phase or upon starvation and play a role in the nutrient-stress response (11). In Bacillus subtilis only three Csp genes were identified (CspB, CspC, and CspD) (12). Previous report has shown that Streptomyces clavuligerus (13) contains SC.7.0 protein homologous to CspA of E. coli. Using antibodies raised against proteins CspB, CspC, and CspD of B. subtilis, we detected in S. aureofaciens five cross-reacting proteins. These proteins are similar in size and were identified in vegetative cells. CspB and CspD are cold inducible. The data presented here indicate that ribosomes having a highly cooperative structure are also a potential target for control mechanisms that generate signals and activate adaptive regulons or developmental programs. In the present paper we set out to determine how streptomycetes respond to downshift in temperature and to the presence of tetracycline, inducing coldshock response. Production of tetracyclines in S. aureofaciens occurs upon exhaustion of inorganic phosphate from cultivation medium. Our data show that cold acclimation and production of tetracycline is accompanied by increased synthesis of Csp and their association with ribosomes.

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MATERIALS AND METHODS Materials. Microbial strain and cultivation: Aerial spores of Streptomyces aureofaciens 84/25 MBU (14) were used to inoculate medium containing (g/l) (NH 4) 2SO 4 2.0, MgSO 4 7H 2O 0.2, CaCO 3 1.0, Yeast extract 2.0, Casamino acids 10.0, glycerol 10.0 and KH 2PO 4 1.0, pH 7.2. After 20 h cultivation at 28°C, cells were used for inoculation of fresh medium. Cells from various stages of growth were harvested by centrifugation and washed with standard buffer containing 20 mM Tris–HCl, pH 7.6, 10 mM MgCl 2, 40 mM NH 4Cl, 6 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride (PMSF). Protein synthesis during cold shock. Streptomyces aureofaciens cells were grown in complete medium at 28°C (optimum growth temperature) to the exponential phase (14 h cultivation). The culture was then divided into four identical portions (60 ml) in 500 ml flasks. Two flasks were shifted to 12°C and incubated at this temperature in the presence or absence of rifamycin (400 mg/ml) for 4 h. Proteins were radiolabeled by adding 100 mCi of trans [ 35S]methionine (specific activity 37 TBq/mmol 21) for the last 30 min of incubation at 12°C. After cultivation cells were harvested by centrifugation at 10,000g for 10 min at 4°C and washed with standard buffer. Cells were disrupted and homogenates were extracted with standard buffer. Cell debris and membranes were removed by centrifugation at 30,000g for 30 min (S30 fraction). Supernatant solution was layered over 20 ml cushions containing standard buffer with 15% sucrose. After centrifugation at 150,000g for 15 h and 4°C in 50.2 Ti rotor (Beckman) supernatant was removed (S150 fraction) and crude ribosomes were washed with 1 M NH 4Cl for 2 h in ice bath. Aggregates were removed by low speed centrifugation and ribosomes were sedimented at 150,000g for 3 h and 4°C. Supernatant solutions (associated proteins with ribosomes) were frozen in liquid nitrogen and stored at 270°C. For electrophoretic analysis, proteins were precipitated with 5% TCA, washed with acetoneether at 220°C. Proteins were analyzed in high resolution two-dimensional system IEF–PAGE (15). Radioactivity of proteins was monitored by autoradiography or by Phosphor-imager. Immunodetection proteins of Csp family. Western blots of twodimensional gels containing Csp proteins were incubated with 5% serum albumin in TBST (20 mM Tris–HCl, pH 7.6, 137 mM NaCl, 0.2% Tween 20) overnight at 4°C. Proteins of the Csp family were detected using antibodies raised against CspB, CspC and CspD of Bacillus subtilis (gift from M. A. Marahiel, Philipps-Universitat Marburg). Blots were incubated with first antibodies in TBST containing 5% serum albumin at room temperature for 1 h. Secondary antibody (rabbit Ig, horseradish peroxidase linked whole antibody from sheep) were incubated with blots in TBST for 1 h at room temperature. ECL Western blotting reagents (Amersham) were used for the detection. Pulse labeling experiments. Rate of protein synthesis was estimated using pulse-label experiments. Samples (5 ml) were pulse labeled for 5 min with [U- 14C]phenylalanine (350 mCi/mol). One-ml samples were precipitated with 1 ml 10% trichloroacetic acid (TCA) and incubated at 90°C for 20 min. TCA precipitates were collected on membrane filters and washed, and radioactivity was measured.

FIG. 1. Autoradiograms of proteins of cells exposed to cold shock or tetracycline. Cells were labeled for the last 30 min with 100 mC/ml trans-[ 35S]methionine. Cell-free extracts (S30 fractions) were analyzed by two-dimensional electrophoresis (MiniProtean II System). The first dimension was carried out in mixture of ampholines (1.6% pH 5–7, 0.4% pH 3–10) and second dimension in 12.5% polyacrylamide gel. Radioactivity of dry gels was monitored by autoradiography. Expression of proteins before cold shock (A) and after incubation at 12°C for 4 h (B). Expression of proteins after incubation of cells from mid log phase of growth in the presence of 200 mg/ml of tetracycline at 28°C for 4 h (C).

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FIG. 2. Immunological detection of Csp proteins. Cells from exponential phase of growth were incubated at 12°C for 4 h and cell-free extract (S30 fraction) was analyzed by IEF/SDS–PAGE using the Investigator system (Oxford GlycoSystem). Second dimension was performed in 15% polyacrylamide gel (20 cm 3 20 cm) and proteins were silver stained (A). Part of gels containing proteins of 5–15 kDa and pI 4 –5 were cut and Western blots probed with antibodies raised against Bacillus subtilis CspB (B), CspC (C) and CspD (D). Second antibody (rabbit Ig) and ECL Western blotting reagents (Amersham) were used for the detection. Assays. Concentrations of phosphate and tetracycline were assayed as described previously (16).

RESULTS When cells of Streptomyces aureofaciens from exponential phase of growth at 28°C (doubling time 4 h) were cooled to 12°C, growth was stopped and after 10 h lag phase growth continued with a doubling time of 28–30 h. We examined expression of proteins after 4 h incubation of cells from exponential phase at 12°C. Cells were labeled for the last 30 min with trans[ 35S]methionine, proteins were analyzed on 2-D gels and radioactivity monitored by autoradiography. The results show that at the middle of a lag phase (after 4 h at 12°C) the shift down in temperature involves transient repression of polypeptide synthesis accompanied by synthesis of at least 32 proteins (Figs. 1A and 1B). To determine whether expression of proteins during a cold induced lag phase is dependent on transcription, cells were incubated for 4 h at 12°C in the presence of rifamycin (400 mg/ml) and labeled for 30 min with [ 35S]methionine. Results of this experiment

showed that rifamycin inhibited incorporation of labeled methionine into proteins thus indicating that expression of protein after 4 h incubation at 12°C is controlled at the transcription level (data not shown). We investigated the effect of subinhibitory concentration of tetracycline on expression of proteins in tetracycline producing strain of Streptomyces aureofaciens. The culture from mid-log phase of growth was incubated at 28°C for 4 h in the presence of 200 mg/ml of tetracycline and cells were labeled for last 30 min with trans[ 35S]methionine. In sensitive cells of E. coli, tetracycline in a concentration of 0.8 –1.0 mg/ml produced a 70% growth inhibition, blocked expression of heat shock proteins (e.g., GroEL, DnaK) and induced a set of proteins including ribosomal proteins and several translation factors (2). Cultures of S. aureofaciens from exponential phase of growth are resistant to more than two orders of magnitude higher concentration of the drug than that of E. coli. The presence of tetracycline (200 mg/ml) resulted in synthesis of 27 novel proteins (indicated by arrows) and 17 upregulated pro-

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FIG. 3. Association of Csp proteins with ribosomes. Ribosomes from cells before and after cold shock or after incubation with tetracycline (200 mg/ml) were isolated as described under Materials and Methods. Ribosomes were washed with 1 M NH4Cl and sedimented by ultracentrifugation. Ribosomal washes were analyzed by two-dimensional electrophoresis and silver stained. Proteins of S30 fraction after cold shock (A). Proteins of ribosomal wash after cold shock in absence (D) or presence of rifamycin (400 mg/ml) (B). Proteins from 14-h-old cells incubated in the presence of 200 mg/ml of tetracycline for 4 h at 28°C (C) Equal amount of proteins (50 mg) were applied on first-dimensional gels.

teins migrated as spots with coordinates identical with those of proteins expressed upon a cold shock (Fig. 1C). Using comigration of samples with purified proteins, detection of protein on Western blots with specific antibodies and microsequencing, several proteins were identified: polynucleotide phosphorylase [1], elongation factors EF-G [7], EF-Tu [13] initiation factor IF-2 [6], b subunits of RNA polymerase [b]. Cold shock as well as tetracycline block expression of proteins DnaK[K] and GroEL[G] (Figs. 1B and 1C). Since little is known about the Csp protein family in streptomycetes, protein extracts (S30 fractions) from cells exposed

to cold shock were resolved on 2-D gels (Fig. 2A) and Western blots containing proteins of 5–15 kDa were probed with antibodies raised against proteins CspB, CspC, and CspD of Bacillus subtilis. Immunodetection analysis reveals five cross-reactive proteins. Antibody against CspC recognized CspB, CspC, CspD and two not yet identified proteins of Csp family (CspX and CspY) (Fig. 2C) while antibodies against CspB (Fig. 2B) and CspD (Fig. 2D) exhibited specificity only for the corresponding protein. To characterize more precisely protein CspB, tryptic peptides of the protein excised from 2D gel were analyzed. One of the frag-

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tetracycline and at 48 h tetracycline concentration reached 400 mg/ml. Cells from 48 h of cultivation were labeled for 30 min with trans[ 35S]methionine and then proteins from ribosomal washes were analyzed on 2-D gels. The data in Fig. 5 represents a part of radioactivity pattern of proteins from cells after 48 h of cultivation, which demonstrate that proteins CspB, CspC, CspD, and CspX are expressed during tetracycline biosynthesis. DISCUSSION

FIG. 4. Kinetics of protein synthesis and changes in inorganic phosphate concentration accompanying transition from primary to secondary metabolism in tetracycline producer S. aureofaciens. Kinetics of protein synthesis (F). Utilization of inorganic phosphate (E). Production of tetracycline (■).

ments contains highly conserved sequence KGFGFIEK that showed 87% identity with the cold shock protein SC7. 0 of S. clavuligerus (13) or CspB of Bacillus subtilis. The fragment is a part of b2 strand of Csp and possesses RNA-binding motif (10). Experiments were performed to examine whether Csp proteins are associated with ribosomes. Cells from mid-log phase were incubated at 12°C for 4 h in the presence or absence of rifamycin and protein extracts were fractionated by ultracentrifugation as described under Materials and Methods. Ribosomal washes with 1 M NH 4Cl were analyzed by 2-D gel electrophoresis. As can be seen from Csp protein patterns of the S30 fraction and ribosomal wash of cells after temperature shift down (Figs. 3A and 3D, respectively) most Csp’s are associated with ribosomes. Densitometric record of Csp spots from ribosomal washes showed that cells incubated in the absence of rifamycin (Fig. 3D) contained about six times more CspB and CspD than those incubated in the presence of rifamycin (Fig. 3B). These results also demonstrate that the amount of CspB and its association with ribosomes increased in the cells incubated in the presence of tetracycline (200 mg/ml) (Fig. 3C). Members of Csp family in E coli are involved in many cellular processes or are required under several physiological conditions (17). We examined whether proteins of Csp family are expressed during production of tetracycline. Transition from primary metabolism to secondary metabolism in S. aureofaciens take places after rate of protein synthesis reaches its maximum and inorganic phosphate is exhausted (Fig. 4). Cells from 24 h cultivation synthesized about 50 mg/ml of

Proteins of Csp family are able to interact with RNA secondary structures formed intermolecularly or intramolecularly and affect RNA folding (5). Binding of Csp to RNA is cooperative and no specific RNA sequences for Csp binding were identified. In addition to well-documented function of Csp in prevention of secondary structure formation in mRNA (18, 19), Csp can bind to single stranded rRNA and stabilize ribosomes in active conformation during temperature shift. Our present finding demonstrating association of Csp proteins (CspB, CspC, CspD and CspX) of S. aureofaciens with ribosomes also supports this suggestion. Protein CspB and CspD were synthesized during the acclimation phase, because in the presence of rifamycin expression of CspB CspD and other proteins in cells from the acclimation phase was blocked.

FIG. 5. Autoradiogram of Csp proteins from ribosomal wash of 48-h-old cells producing tetracycline. Cells were labeled for 30 min with trans[ 35S]methionine and ribosomal wash was analyzed by IEF/ SDS–PAGE. Second dimension was performed in 15% polyacrylamide gel. Part of the gel containing Csp proteins was dry and radioactivity was monitored by autoradiography.

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Recently, the effect of kanamycin and chloramphenicol on the inducibility of CspA, CspB, and CspG of E. coli was examined (20). The synthesis of all these proteins was induced virtually in the absence of synthesis of any other proteins, indicating that the cold shock proteins are able to bypass inhibitory effects of the antibiotics. In our previous studies (21, 22) we have demonstrated that purified (1 M NH 4Cl washed) tightcoupled ribosomes of S. aureofaciens are more sensitive to tetracycline then ribosomes of E. coli in the PhetRNA binding and elongation factor dependent translation of poly(U). Two-dimensional gel analysis revealed that the presence of subinhibitory concentration of tetracycline (200 mg/ml) stimulate protein synthesis of the cells from exponential phase of growth including cold inducible proteins. These data indicate that additional proteins associated with ribosomes are required for polypeptide synthesis in the presence of the drug. The high level of tetracycline resistance in S. aureofaciens may not be explained only by a blockage in transport of the antibiotic or by overexpression of Tet(M) or structurally similar proteins which compete with EF-G for ribosomal binding sites, since high levels of Tet(M) inhibit protein synthesis and appear to form stable complexes (23). According to the “Cold-shock ribosome adaptation model” (7) an increase in nontranslatable ribosomes induced by cold or tetracycline can be a signal for induction of cold shock response. At low temperature or in the presence of high levels of tetracycline ribosomes have a limited capacity to initiate translation of most mRNAs and initiate translation of certain types of mRNA for proteins which are able to reactivate ribosomes and/or for enzymes of secondary metabolism. In E. coli ribosomal protein S1 is essential for correct positioning of the mRNA into the decoding site on ribosomes. The structure of the S1 domain is very similar to that of cold shock proteins suggesting that they are both derived from an ancient nucleic acid-binding protein (24). We have found previously (25) that functionally active protein to S1 of E. coli is absent in S. aureofaciens. Streptomycetes contains many mRNA lacking Shine-Dalgarno sequences (26 – 29) including leaderless transcripts encoding essential proteins that confer antibiotic resistance. Some of these leaderless mRNAs carry sequences (downstream box) which are complementary to 16S RNA nucleotides 1469 –1483 (anti-downstream box). As expression of Csp proteins and their association with ribosomes differ during development or temperature shift down these proteins can be considered as trans-acting protein factors with possible S1 function to form contacts with the coding region of leaderless mRNAs. Additional experiments using defined systems are required to test this possibility.

ACKNOWLEDGMENTS We thank M. A. Marahiel for providing antibodies. This work was supported from the Grant Agency of the Czech Republic (203/98/0422).

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